REPORT SHEET

ANALYSIS OF PROTEINS

DATA AND OBSERVATIONS

A. Calculate the percentage of casein in the milk

% casein= weight of solid casein/ 50g of milk x 100

Computation:

Weight of solid casein: 1.1g

% casein = 1.1g / 100g x 100 = 1.1%

B. Test for Proteins

1. Biuret Test

Solutions Observation/Positive Color Reaction

For the first test tube, a 2mL solution of Biuret reagent was
combined with casein solution, and after 2 minutes, the
Test tube 1 combination was light purple in color. The casein solution tests
positive for proteins in this assay.
Casein suspension
The second test tube contains 2mL of Biuret reagent mixed with
2% glycine. Blue hue was noticed after 2 minutes of relaxation.
Test tube 2 As a result, 2% glycine has no proteins.

2% glycine
In a test tube, a 2mL solution of Biuret reagent was mixed with
2% gelatin. After 2 minutes, the hue shifted to purple, indicating
Test tube 3 the presence of proteins.

2% gelatin
For the first test tube, a 2mL solution of Biuret reagent was
combined with 2% albumin, and the liquid became purple after
 2 minutes. Albumin is shown to contain proteins in this assay.
Test tube 4

2% albumin
For the first test tube, a 2mL solution of Biuret reagent was
combined with 2% albumin, and the liquid became purple after
Test tube 5 2 minutes. Albumin is shown to contain proteins in this assay.

1% tyrosine

2. Hopkins-Cole Test

Solutions Observation/Positive Color Reaction

Test tube 1 After shaking it after adding 0.5 mL of Hopkins-Cole reagent,
proceed as directed. For example, when dropwise conc. H2SO4
Casein suspension was added, the hue changed to red-orange and a purple ring was
seen, highlighting the two layers.
Test tube 2 The color was clear once the process was completed, and no
purple ring was observed, which is interpreted negative.
2% glycine
Test tube 3 Once the procedure was done, the color was bright and hazy, with
no purple ring visible, which is viewed negatively.
2% gelatin
Test tube 4 Once the method is done, 0.5 of Hopkins-Cole reagent is added
and shaken. Furthermore, when dropwise conc.H2SO4 was
2% albumin added, the hue changed to violet and purple.
Test tube 5 (After the procedure was done, the color was clear, and no purple
ring was detected, which is interpreted negatively.)
1% tyrosine
3. Millon’s Test

Solutions Observation/Positive Color Reaction

A boiling water bath was used to heat the casein solution and
Millon's regent combination for 3 minutes. The tint turned to
Test tube 1 light yellow with a clous-like pattern on top.

Casein suspension
The test tube containing a combination of Millon's reagent and
2ml of glycine was colorless before heating, but the color did not
Test tube 2 change after heating. Glycine fails the test because it does not
contain a phenol functional group that attaches to the side
2% glycine chain.

Test tube 3 Gelatin has the same hue as glycine—it is colorless. Gelatin
yields a negative result because it lacks the presence of phenol-
2% gelatin containing chemicals.

Test tube 4 After 3 minutes of heating, the mixture has the color of clouds.
Albumin is bad since it should be brick red in color.
2% albumin
The combination is a clear solution before heating, and the color
stayed the same after 3 minutes of heating in a water bath.
Test tube 5 Tyrosine does well in the test because it includes a phenol
functional group.
1% tyrosine

4. Ninhydrin Test

Solutions Observation/Positive Color Reaction

Saturated Casein

Test tube 1 After boiling the solution containing saturated casein

Casein suspension and 0.2% ninhydrin for many minutes, there was no
change in its color or consistency. It remained
transparent, indicating the lack of proteins.

Extracted casein

After several minutes of heating the solution

containing extracted casein and 0.2% ninhydrin, a
little change in color was detected, as it became a
blend of white with a hint of purple. Thus, there are
proteins present.
It was found that the solution containing 2% glycine
and 0.2% ninhydrin became purple after being heated
Test tube 2
for several minutes. Therefore, this shows the

2% glycine existence of proteins.

Test tube 3 After boiling the solution containing 2% gelatin and

0.2% ninhydrin for many minutes, its color and
2% gelatin
consistency remained unchanged. It remained
transparent, suggesting that there are no proteins
present in the solution.
It was found that the solution containing 2% albumin
and 0.2% ninhydrin became purple after being heated
Test tube 4
for several minutes. Therefore, this indicates the

2% albumin existence of proteins.

After several minutes of boiling a solution containing

2% tyrosine and 0.2% ninhydrin, its color changed to
Test tube 5
purple, indicating the existence of proteins.

1% tyrosine
5. Sulfur Tests

Solutions Observation/Positive Color Reaction

Negative Result - (Following the addition of 1 ml of 3M
NaOH and 15 minutes of boiling,) There has been no
Test tube 1
formation of a black precipitate in Test Tube 1.

Casein suspension
Negative Result - After adding 1ml of 3M NaOH and
boiling for 15 minutes, no black precipitate developed
Test tube 2
in Test tube 2.

2% glycine
Negative Result - After adding 1 ml of 3M NaOH and
boiling for 15 minutes, no black precipitate has
Test tube 3
developed in Test tube 3.

2% gelatin

Test tube 4 Positive Result - After adding 1 ml of 3M NaOH and

boiling for 15 minutes, no black precipitate has
2% albumin
developed in Test tube 3. However, no black
precipitate forms in Test tube 4, but damp lead
acetate paper becomes brown.
 Test tube 5 Negative Result - After adding 1ml of 3M NaOH and
boiling for 15 minutes, no black precipitate has
1% tyrosine
developed in Test tube 55.

Test tube 6 Positive Result - Test tube 6 has no precipitate after

adding 1 ml of 3M NaOH and boiling for 15 minutes,
Extracted casein
but wet lead acetate paper becomes dark.reflecting an
unfavorable outcome (proteins are absent)

6. Xanthoproteic Test

Solutions Observation/Positive Color Reaction

While whirling, 10 drops of concentrated HNO3 were
added to the casein solution in a test tube. After
Test tube 1
putting the combination in a hot water bath for one
minute, it was determined that the Casein suspension
 failed the Xanthoproteic test due to the lack of a
Casein suspension
solution with a dark yellow or orange hue.
While whirling, 10 drops of concentrated HNO3 were
introduced to 2% glycine in a test tube. After putting
Test tube 2
the combination in a hot water bath for one minute, it

2% glycine was determined that 2% glycine failed the

Xanthoproteic test due to the lack of a solution with a
dark yellow or orange hue.
While whirling, 10 drops of concentrated HNO3 were
added to the 2% gelatin in a test tube. After putting
Test tube 3
the combination in a hot water bath for one minute, it

2% gelatin was determined that 2% gelatin failed the

Xanthoproteic test due to the lack of a solution with a
dark yellow or orange hue.
While whirling, 10 drops of concentrated HNO3 were
added to the 2% albumin in a test tube. After putting
Test tube 4
the combination in a hot water bath for one minute, it

2% albumin was determined that 2% albumin is negative for the

Xanthoproteic test due to the lack of a solution with a
dark yellow or orange hue.
While whirling, 10 drops of concentrated HNO3 were
added to the 2% tyrosine in a test tube. After putting
Test tube 5
the mixture in a hot water bath for one minute, a

1% tyrosine dark yellow or orange-colored solution was detected

in the test tube, indicating that 2% tyrosine is positive
for the Xanthoproteic test.

C. Denaturation Tests

Solutions Observation/Appearance after Denaturation

 Saturated Casein

Casein suspension There were no visible changes in the appearance of

the solution after boiling. It retained its transparency.

Extracted Casein

There was no change in the solution's appearance

after boiling, but a beige precipitate developed at the
bottom.
Casein suspension + Saturated Casein
ethanol
After 3-4 minutes of shaking the fluid, there were no
changes. Its hue and texture remained transparent
and clear

Extracted Casein

After 3 to 4 minutes of vigorous shaking, the color of

the solution changed from hazy to transparent, and a
cloud-like precipitate formed, part of which floated
while the rest sunk to the bottom.
Saturated Casein

Test tube 1 After adding a few drops of 5% lead acetate, the

solution remained transparent even after 8 minutes,
Casein suspension +
indicating that no additional modifications or
lead acetate
formations had occurred..

Extracted Casein
 After adding a few drops of 5% lead acetate, the
consistency and color of the solution remained
unchanged, and only a beige precipitate was detected
sinking to the bottom. After around 8 minutes, the
hue changed from faded to clear, and cloud-like
precipitation formed at the bottom beside the beige
precipitation.
Saturated Casein

Test tube 2 After adding a few drops of 5% mercuric chloride, the

color remained transparent and cloudy precipitate
Casein suspension +
formed. A few minutes later, the cloud-like
HgCl2
precipitation descended to the ground.

Extracted Casein

Texture and color remained when a few drops of 5%

mercuric chloride were added. Even after a few
minutes of observation, no modifications or
formations were detected.
Saturated Casein

Test tube 3 Following the addition of a few drops of 5% ferric

chloride, the solution became yellow. Other than this,
Casein suspension +
no alterations or formations were detected.
FeCl3

Extracted Casein

Following the addition of a few drops of 5% ferric

chloride, the solution became yellow. Other than this,
no alterations or formations were detected.
Questions:

1. Explain why casein precipitates when acetic acid is added to it?

Casein production goes increased with acetic acid content. For the sake of
simplification, acetic acid may hydrolyze protein bonds into peptide bonds.
This modification improves casein yields by increasing the solubility of simple
proteins in water.

2. Cite five examples of denaturation of proteins encountered in everyday

life.

1. Digestion
2. Wiping alcohol
3. When milk curdles.
4. Boiling eggs
5. Perming hair

3. Why is milk or raw egg used as antidote in cases of heavy metal ion
poisoning?

Proteins in milk and raw egg contain sulfhydryl (-SH) groups that may bind
heavy metal ions and flush them out of the body, which is why they are
employed as an antidote for heavy metal poisoning. In detail, the egg whites
will absorb the metal while the milk will neutralize the stomach acid, causing
the heavy metal to be vomited out of the body. In conclusion, raw egg or milk is
used as an antidote to coat the lining of the mucous membranes in situations
of heavy metal ion poisoning. It provides a barrier against the heavy metals,
delaying their absorption.